Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by mutations in the dystrophin gene. Despite years of study, the mechanism of muscle cell destruction resulting from dystrophin deficiency remains unclear. Dystrophin links the subsarcolemmal cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC). Dystrophin is thought to be important for mechanical stability of the sarcolemma; however, it has become clear that portions of the DGC also play signaling roles. Signaling components of the DGC include the peripheral membrane proteins nitric oxide synthesis (nNOS), the syntrophins (SYN) and the dystrobrevins (DB). Important questions remain as to how these peripheral components are assembled, and how this assembly modulates their role in preventing dystrophy. In addition, the histopathology of muscles from DMD patients and the mdx mouse model is associated with focal areas of myofiber necrosis and a significant inflammatory response. These observations have generated controversy as to the relative importance of mechanical, signaling and immune-mediated mechanisms of muscle fiber death. Increased knowledge of the organization and function of the DGC will be critical for effective development of a therapy. From a gene therapy perspective, dystrophin delivery leads to re-assembly of the DGC and halts further mechanical damage, but previously activated immune effector cells or altered signaling pathways might prevent a full functional rescue. Effective therapy might therefore require delivery of dystrophin before significant pathological abnormalities become established. This application proposes to address the relationship between improper assembly of the DGC and the mechanical, signaling and immune-mediated mechanisms of muscle cell destruction. The specific aims will test the following hypotheses: 1) the peripheral portion of the DGC is functionally tethered to the sarcolemma by an interaction between dystrobrevin and the sarcoglycans; 2) the inflammatory response seen in young mdx mice results primarily from the loss of mechanical protection to the sarcolemma; and 3) the DGC can be functionally assembled both in utero and in young mice. These studies will address the organization of the major signaling center of the DGC. The mechanical, signaling and immune mediated mechanisms of pathology will be separated to enable assessment of their individual contributions to dystrophy. Finally, studies will be performed to compare the effectiveness of early vs. late DGC assembly in halting disease pathology. Successful completion of these goals will lead to a better understanding of DMD pathology and improve the prospects for a treatment.